Molecular Basis for\nShort-Chain Thioester Hydrolysis\nby Acyl Hydrolases in <i>trans</i>-Acyltransferase Polyketide\nSynthases

Polyketide synthases (PKSs) are multidomain enzymatic\nassembly\nlines that biosynthesize a wide selection of bioactive natural products\nfrom simple building blocks. In contrast to their <i>cis</i>-acyltransferase (AT) counterparts, <i>trans</i>-AT PKSs\nrely on stand-alone ATs to load extender units onto acyl carrier protein\n(ACP) domains embedded in the core PKS machinery. <i>Trans</i>-AT PKS gene clusters also encode stand-alone acyl hydrolases (AHs),\nwhich are predicted to share the overall fold of ATs but function\nlike type II thioesterases (TE<sub>II</sub>s), hydrolyzing aberrant\nacyl chains from ACP domains to promote biosynthetic efficiency. How\nAHs specifically target short acyl chains, in particular acetyl groups,\ntethered as thioesters to the substrate-shuttling ACP domains, with\nhydrolytic rather than acyl transfer activity, has remained unclear.\nTo answer these questions, we solved the first structure of an AH\nand performed structure-guided activity assays on active site variants.\nOur results offer key insights into chain length control and selection\nagainst coenzyme A-tethered substrates, and clarify how the interaction\ninterface between AHs and ACP domains contributes to recognition of\ncognate and noncognate ACP domains. Combining our experimental findings\nwith molecular dynamics simulations allowed for the construction of\na data-driven model of an AH:ACP domain complex. Our results advance\nthe currently incomplete understanding of polyketide biosynthesis\nby <i>trans</i>-AT PKSs, and provide foundations for future\nbioengineering efforts to offload biosynthetic intermediates or enhance\nproduct yields.